Nucleosides play significant roles in different body systems, including the nervous and the vascular systems. Their specific functions in these systems is determined by the presence of purine and pyrimidine receptors which react with triphosphate nucleosides and their derivatives at the surface of numerous cell types. The presence in the vascular system of both ectoATPase and ectoADPase activities, that cleave tri- and diphosphate nucleosides respectively, has been known for many years. By their location and kinetic properties, these different catalytic activities could influence these systems. Both activities were previously attributed to two distinct enzymes. These activities were characterized and showed that, in bovine aorta, a single enzyme was responsible for the sequential hydrolysis of ATP and ADP.
The enzyme responsible for that sequential cleavage of the γ and β phosphate residues of polyphosphorylated nucleosides are commonly called nucleoside triphosphate diphosphohydrolases (NTPDase) or apyrases (EC 3.6.1.5). These enzymes are generally activated in the presence of divalent cations Ca+2 or Mg+2, while sodium azide inhibits their activity azide is an inhibitor of NTPDase1 and a few other NTPDase but not all of them. The catalytic site of these enzymes is generally exposed to extracytoplasmic spaces (ectoenzymes).
In plants, NTPDase enzymes are found in the cytoplasm, in soluble or membrane-associated forms, and are generally more active in acidic conditions. Although their precise function remains unknown, some evidences suggest their implication in the biosynthesis of carbohydrates. At the opposite, the activity of NTPDases is higher at neutral or alkaline pH in invertebrates species, where they principally shown to act as antihemostatic agents in saliva and in salivary glands of hematophagous insects.
In vertebrates, a limited number of studies have already defined a diversity of NTPDases. The first mammalian NTPDase has been isolated from pancreas and was further identified in other tissues, including bovine aorta. It is recognized in the art that NTPDase may be the object of other nomenclature. For exaple, NTPDase1 may refers to vascular ATPDase or CD39.
Nucleosides and thus, NTPDases, contribute to the vascular system homeostasis. Extracellular nucleosides present in the blood due to, e.g., arterial vascular injury can influence cardiac function, vasomotor responses, inflammatory processes, thrombosis, and platelet activation. To maintain blood fluidity and flow, the normal vascular endothelium inhibits coagulation and platelet activation and promotes fibrinolysis. Quiescent endothelial cells are considered to directly express natural anticoagulants and thromboregulatory factors, therefore preventing thrombosis, which usually develops as a consequence of overwhelming these antithrombotic mechanisms. This may occur following the heightened production of locally produced mediators, including cytokines, activated complement components and particularly extracellular nucleotides. In the bovine aorta, NTPDase1 was found to be associated with smooth muscle cells and endothelial cells and could inhibit ADP-induced platelet aggregation. It was further showed that concurrent addition of a semi purified fractin of NTPDase and ATP to platelet-rich plasma resulted in an immediate dose-dependent platelet aggregation caused by the accumulation of ADP, followed by a slow desaggregation attributable to its hydrolysis into AMP. In the absence of NTPDase, ATP does not induce any aggregation while ADP initiates an aggregation which extent is limited by the ADPase activity of the enzyme.
Mechanism of nucleosides actions in blood vessels implicates, between others, the binding to and stimulation of purinergic/pyrimidinergic type-2 (P2) receptors. This stimulation P2Y receptors initiates G protein-coupled signaling pathways and results in activation of platelets, endothelial cells (ECs), monocytes/macrophages, and leukocytes and could culminate in vascular thrombosis and inflammation in vivo. ATP and ADP regulation of platelet aggregation appear to occur through the concomitant activation of platelet P2Y1 and P2Y12 receptors. Indeed ADP is a major platelet recruiting and activating factor, whereas ATP acts as a weak competitive antagonist of ADP for platelet P2 receptors. This latter protective action of ATP may limit the formation of intravascular platelet aggregation and help localize thrombus formation to areas of vascular damage. NTPDases also attenuate the aggregation elicited collagen and low level of by thrombin but not by the platelet activating factor (PAF), the first two agonist effects being caused by a release of platelet ADP. It has therefore been suggested that NTPDase had a dual role in regulating platelet activation. By converting ATP released from damaged vessel cells into ADP, the enzyme induced platelet aggregation at the sites of vascular injury. By converting ADP released from aggregated platelets and/or from hemolyzed red blood cells to AMP, the NTPDase could inhibit or reverse platelet activation, and consequently limit the growth of platelet thrombus at the site of injury.
Platelets are particles found in whole blood that initiate and provide the structural basis for the haemostatic plug necessary to stop bleeding. Platelets depend on adhesive interactions with extracellular proteins and other cells for proper function. The external platelet plasma membrane surface is covered with a variety of membrane bound glycoproteins, many of which have adhesive functions. Perhaps the most abundant platelet membrane adhesive proteins belong to the integrin superfamily which include the glycoproteins; GP Ib IIIa, GP Ia IIa, GP Ic IIa, GP Ib IX, and the fibronectin and vitronectin receptors. Each integrin receptor is an heterodimer displaying characteristic affinity and specificity toward various extracellular matrix proteins such as; von Willebrand factor (vWF), collagen, entactin, tenascin, fibronectin (Fn), vitronectin (Vn), and laminin, as well as fibrinogen (Fg) and thrombospondin. The most abundant integrin found on normal platelet surfaces is GP IIb et GPIIIa comprising about 50,000 molecules per platelet, representing about 2% of the total platelet protein. GP IIb IIIa is a non-covalent, calcium ion dependent heterodimer complex and restricted in distribution to platelets and other cells of the megakaryocytic lineage. On activated platelets, GP IIb IIIa binds a number of adhesive proteins with varying affinities; fibrinogen, fibronectin, von Willebrand factor, vitronectin and thrombospondin. It is believed the most important interactions mediating platelet aggregation involve GP IIb IIIa binding with the trinodular fibrinogen and, to a lesser extent, with the filamentous von Willebrand factor.
Platelets are key components of all blood clots propagating within the arterial circulation and thus are an obvious therapeutic target in attempts to inhibit coronary artery thrombosis. Despite currently available therapies, a significant number of ischemic events, such as myocardial infarction, stroke, and death, occur each year. These events are generally the result of blood clots blocking the arteries supplying oxygen to heart or brain tissue. Therefore, there exists a need for therapeutics that effectively regulate platelet activation for the purpose of controlling platelet aggregation.
Angiogenesis is also a highly complex multistep phenomenon that incorporates both formation of new capillaries and expansion or extension of existing blood vessels. An associated increased permeability to plasma solutes results in the deposition of a provisional matrix in which fibrin is a major component. New vessel growth may be modulated by monocyte/macrophages that secrete angiogenic factors and metalloproteases that facilitate endothelial cell migration. Supporting cells are also essential for new vessel growth and angiogenesis, for example, smooth muscle cells in vascular maturation and arteriogenesis and pericytes in the protection of newly developing endothelial cell-lined tubes from rupture and regression.
In this context, UTP has been shown to be mitogenic and chemotactic for endothelial cells in vitro. Interestingly, binding of angiostatin, a proteolytic fragment of plasminogen and potent antagonist of angiogenesis, to ATP synthase expressed on endothelial cells, has been shown to mediate antiangiogenic effects.
To decipher the mechanisms of such interactions, the role of nucleotides in angiogenesis cd39-null (or Entpd1)mouse model, in which aberrant regulation of nucleotide P2 receptors has been observed, was investigated. CD39 (also referred to as nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) was shown to be the major vascular endothelial membrane ectonucleotidases and to hydrolyse nucleoside triphosphates and diphosphates, ultimately to the nucleoside analogues; these products having mitogenic effects on endothelial cells in vitro. Because angiogenesis is critical to the progression of various diseases, for example, cancer, rheumatoid arthritis, and diabetic retinopathy, there exists a need for compounds capable of preventing or reducing angiogenesis in patients suffering from an angiogenesis-associated condition. NTPDase1/CD39 could now be used to reduce platelet aggregation, thrombogenicity as well as angiogenesis. However, the solubilization of the protein actually lead to a decrease in nucleoside phospohydrolase activity. It would thus be highly desirable to be provided with a stable NTPDase for the treatment of prevention of diseases related to blood clotting or angiogenesis.
While some products related to ectohydroxynucleotides as described before exist in the art, there is still place for new molecules allowing platelet aggregation modulation and/or control in different physiological normal or pathological conditions.